Our goal is to speed standardization of sample sparing assays of immune function through the design and validation of a flexible microfluidic system that is capable of automated processing and multi-parameter analysis of rare cells. A novel cell capture mechanism (Vortex cell trapping) is utilized to isolate rare cells into nanolier arrays, and fully automate the steps involved in profiling cell function (i.e., activation, incubaton, labeling). We utilize the device to perform sample sparing versions of two important assays of immune function: the CD154 and peptide-MHC tetramer assay, and investigate the effect of common processing steps on the detection limits and variability at input sample volumes that are beyond the limits of existing methods. Unlike current microfluidic systems, our approach enables direct capture of cells from complex clinical samples, isolation and monitoring of antigen-specific cells, and rapid exchange of buffer around the cells for processing without loss. In addition, the microfluidic traps are designed for direct, post-process ejection of the cells to existing multi-parameter analytical platforms (i.e., cell surface marker, transcription level, or TCR sequence analysis) to enable faster translation to clinical labs. The Vortex trap allows direct ex vivo cytokine secretion profiling of rare cells through peptide-MHC tetramer or CD154-based capture. Both of these assays have recently enabled direct monitoring of functional immune response in large-volume clinical samples. However, analysis of antigen-specific cells in volume or cell limited biological specimens remains challenging, and an automated method for ex vivo secretion profiling is desired. As such, we propose to analytically validate our cytokine profiling assay using both a multiple sclerosis (MS) (e.g., clinical need to analyze rare cells with pathogenic secretion profiles), and viral vaccine model (e.g., clinical need to screen functional response of cells from low sample volumes). Clinical validation will be performed using patient-derived samples, and will include direct comparisons to the current standard assays (i.e., T cell cloning or flow-cytometer-based pMHC tetramer or CD154 assays). Our approach is supported by strong preliminary data, and we have assembled a strong academic-industrial partnership consisting of UCLA (Vortex cell trapping), Yale University/Benaroya Research Institute (pMHC tetramers, T cell lines, and patient samples), and GE Global Research (cell-cytokine capture beads, and analytical interface).